We present an easy-to-use, disposable, thermoplastic microwell chip designed to support screening and high-resolution imaging of single-cell behavior in two- and three-dimensional (2D and 3D) cell cultures. We show that the chip has excellent optical properties and provide simple protocols for efficient long-term cell culture of suspension and adherent cells, the latter grown either as monolayers or as hundreds of single, uniformly sized spheroids. We then demonstrate the applicability of the system for single-cell analysis by correlating the dynamic cytotoxic response of single immune cells grown under different metabolic conditions to their intracellular cytolytic load at the end of the assay. Additionally, we illustrate highly multiplex cytotoxicity screening of tumor spheroids in the chip, comparing the effect of environment cues characteristic of the tumor microenvironment on natural killer (NK)-cell-induced killing. Following the functional screening, we perform high-resolution 3D immunofluorescent imaging of infiltrating NK cells within the spheroid volumes.